Laser scanning cytometry is an effective diagnostic tool in determining various characteristics of cells including abnormalities, for the facilitated treatment of diseases and conditions associated with such abnormalities. It is not however designed for actual visual imaging of cell configuration and features. The construction and use of laser scanning cytometers has been described by Kamentsky and Kamentsky (Cytometry, 12:381-387, 1991) and in U.S. Pat. Nos. 4,647,531 and 5,072,382, the disclosures of which are incorporated herein by reference thereto.
In summary, a laser scanning cytometer such as the LSC.TM. cytometer, available from CompuCyte Corp., scans cell specimens, on a microscope slide positioned on a microscope stage, with a laser beam which is oscillating in the Y direction of the microscope stage. Voltage levels from optical detectors are synchronized and digitized to produce a raster of values from captured light. Subsequently, the microscope stage is advanced in the X direction, and the scan digitization is repeated. The cycle is repeated until a two dimensional (x-y) array of measurements is acquired. This two dimensional array is segmented by image processing techniques, and features for cells or objects of interest on the specimen are extracted and stored in a list mode data file.
One of the parameters measured by the LSC is forward light scatter. In this mode of operation, a focused laser beam passes through the specimen slide and laser scattered light is intercepted by a blocker bar before reaching a photodiode detector located beneath the blocker bar. In the reference position where there is no object in the laser beam path, the laser light is entirely prevented from reaching the detector, and the output of the detector is a low (zero) voltage signal. When a cell or other object is in the path of the laser beam, laser light is diverted from its original path, and is scattered over a range of angles (i.e., forward light scattering). A portion of this scattered laser light bypasses the blocker bar and strikes the face of the detector which provides an output signal characteristic of the particular way the light is blocked and scattered.
The output signal from the detector increases proportionally relative to the amount of light scatter. The resulting two dimensional memory array image appears as a dark field image with a black background, with the objects of interest appearing as a generally undefined bright image. There is however no physical image of the actual cells which can be discerned.
Laser scatter and absorption have been used extensively in flow cytometers and with other similar instruments, wherein data from either single or combined sensors are obtained as values for a cell in toto, with no capabilities for cellular imaging and subsequent localization of cellular constituents. Slit scan flow techniques extract subcellular information about cells, but still do not provide images of the cells.